Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP) function and accumulation in the extracellular space of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization that is also one of the natural substrates of TNAP. HPP is characterized by defective mineralization of bones (rickets or osteomalacia), and teeth that display a lack of acellular cementum, dentin and enamel hypomineralization and periodontal defects. During the past funding period, we demonstrated the effectiveness of daily enzyme replacement therapy using mineral-targeted recombinant TNAP (asfotase alfa) to prevent the skeletal and dental defects in the TNAP knockout (Alpl-/-) mouse model of infantile HPP. This therapy is now in phase III clinical trials in patients with life-threatening HPP. These clinical tials are revealing aspects of HPP disease whose pathophysiology we do not yet understand nor improve, such as the premature fusion of skull bones (craniosynostosis) and calcification of the kidney parenchyma (nephrocalcinosis). Patients currently under treatment with asfotase alfa still require craniectomy to relieve intracranial pressure and their nephrocalcinosis has not resolved after five years of treatment. In addition, there is the concern that long-term treatment with asfotase alfa, because of the mineral- seeking properties of this drug, may accelerate the development of vascular calcification in adult patients. Thus, having gone from Bench to Bedside during the last funding period, this competitive renewal application is Back to the Bench. Taking advantage of our collection of mouse models we propose hypotheses-driven specific aims that will ask clinically relevant questions that will help improve the treatment of HPP patients. We will test the hypothesis that changes in purinergic signaling affect the behavior of calvarial cells leading to craniosynostosis. We also surmise that lack of TNAP in the kidney lead to increased tissue insult by endotoxins leading to mesenchymal damage that serves as a nidus for nephrocalcinosis. We will also assess the relative risks-to-benefits of enzyme replacement with mineral-targeted vs soluble non-targeted enzyme. Our work will fill a void in our understanding of what biochemical pathways are affected in soft tissues by the lack of TNAP. Our studies will also provide crucially needed pathophysiological data to help improve the clinical management of patients with life-threatening HPP and point to the most appropriate therapeutic approach for patients with the milder forms of the disease.